Optical microscopy involves the projection of light or radiation onto a sample, and the subsequent detection of reflected, scattered or fluorescence light from the sample.
One example of an optical microscope system is a traditional point scanning confocal microscope. In point scanning confocal microscopy, a single diffraction-limited point of light is projected onto a sample. By imaging that point onto a single element detector, the reflected, scattered or fluorescence light originating from that point in the sample can be measured. A single pinhole placed at a conjugate image plane located between the sample and the detector rejects out of focus light and creates the confocal effect. By scanning the point of light in a manner designed to illuminate the focal plane, for example, by raster scanning, an image of the sample can be constructed point by point. By moving the focal plane optically or by moving the sample, multiple focal planes can be imaged and a 3D image constructed.
The use of optical fibers for light delivery in optical microscopes has been established for many years. Throughout this document, the term “fiber” means “optical fiber”. For traditional point scanning confocal microscopy, the only fiber that can be used effectively for illumination of the sample is a single mode fiber. A single mode fiber is a fiber that is designed for the transmission of a single spatial mode of light as a carrier. This mode of light may contain a variety of different wavelengths, although the range of wavelengths that can be transmitted is a function of the cross-sectional dimensions of the core of the fiber. Typical single mode fibers with cores of circular cross section have core diameters only slightly larger than the wavelengths of light that they transmit. For example, a fiber that transmits in a band around 488 nm has a core diameter of approximately 3.5 μm. Because of the small diameter of the fiber core, single mode fibers are used most often with laser sources. Other sources of radiation are difficult or impossible to couple into single mode fibers with good efficiency.
The cone angle of light that can be coupled into and is emitted from a single mode fiber is related to the numerical aperture (NA) of the fiber. The NA of a single mode fiber is a function of the refractive indices of the fiber core and cladding. The distribution of light emitted from a single mode fiber is well approximated by a Gaussian shape, the width of which is determined by the NA and by the core diameter of the fiber as well as by the wavelength of the light.
The light that is emitted from the distal end of a single mode fiber may be considered equivalent to light that is emitted from a diffraction-limited source. This fiber tip is re-imaged through the pinhole and onto the sample at or near its diffraction-limited size.
FIG. 1 illustrates example optics for projecting light from a distal end 100 of a single mode fiber 102 with a core diameter DF and numerical aperture NAF (related to the illustrated half-angle θ1) to a single pinhole 108 of a diameter DP. The optics include a lens 104 having a focal length F1 and a lens 106 having a focal length F2. The light exits the single mode fiber 102 with a spread of angles given by the numerical aperture NAF of the single mode fiber 102. In general, a numerical aperture NA of a fiber is expressed by Eqn. 1 as:NA=n sin θ,  (1)
where n is a refractive index of the surrounding medium to which the light exits from the distal end of the fiber, exit angle θ is the angle of divergence of light with respect to an optical axis of the fiber, and sin refers to the trigonometric sine function. In the case that the surrounding medium is air, the refractive index n is equal to one, that is n=1. For small angles, θ, and in air, the numerical aperture NA and exit angle θ are approximately equal, that is NA≅θ. The term numerical aperture NA has two definitions when used with fibers. The numerical aperture NA may be defined as a function of the refractive indices of the core and cladding or may be defined as in Eqn. 1. In the ray optics approximation the two definitions are equivalent. In practice, the numerical aperture NA as defined by Eqn. 1 is often less than the numerical aperture NA as defined by the refractive indices of the core and cladding. Throughout this document, the numerical aperture NA is defined by Eqn. 1 unless explicitly noted otherwise. By placing the distal end 100 of the single mode fiber 102 the distance F1 from the lens 104, light passing through the lens 104 is collimated. A diameter of the lens 104 should be large enough to capture the light emitted from the distal end 100 of the fiber 102. By placing the lens 106 at the distance F2 from the pinhole 108, the collimated light incident on the lens 106 is focused by the lens 106 through the pinhole 108, as illustrated by half-angle θ2.
Typically, it is desired in a confocal microscope imaging system for the pinhole spot to be imaged at or near the diffraction limit of the microscope. To produce the minimum imaged spot size, the light transmitted through the pinhole 108 should be the same or larger than the largest numerical aperture NAMS of the microscope as measured at the image plane where the pinhole 108 is located. If the numerical aperture of light exiting the pinhole is larger than numerical aperture NAMS of the microscope, then the minimum imaged spot size can be achieved, however some of the light will be rejected by the microscope optics. Ideally, the numerical aperture of light exiting the pinhole should closely match the numerical aperture NAMS of the microscope so that the optimum resolution and light transmission to the sample can be achieved. The diameter DP of the pinhole 108 should be chosen so that the light exits the pinhole 108 at an angle θ3 given by the numerical aperture NAMS of the microscope as determined by optical diffraction theory. That is, NAMS=n sin θ3.
The focal length F1 of the lens 104 and the focal length F2 of the lens 106 should be chosen to provide an appropriate magnification M of the light exiting the single mode fiber 102 so that the focused light on the pinhole 108 just fills the pinhole diameter DP. For a diameter DF of light exiting the fiber 102, this magnification M is expressed by Eqn. 2 as:
                    M        =                                            D              P                                      D              F                                =                      -                                                            F                  2                                                  F                  1                                            .                                                          (        2        )            
The negative sign indicates that the image of the distal end 100 of the single mode fiber 102 at the plane of the pinhole 108 is inverted. The exact value of the magnification M may be adjusted in practice to fine-tune the trade-off between resolution and light transmission.
In order to achieve both near-diffraction-limited imaging and high light transmission to a sample using a point scanning confocal microscope, the fiber used to deliver radiation from the radiation source to the microscope should be a single mode fiber. If the single mode fiber 102 of FIG. 1 was replaced with a larger diameter fiber, the spot size produced at the plane of the pinhole 108 would be too large to efficiently pass through the pinhole 108. While the magnification M could be reduced to permit the light to efficiently pass through the pinhole 108, for example, by changing one or both of the focal lengths F1 and F2, this would cause a corresponding increase in the numerical aperture of the light exiting the pinhole 108. This mismatch in the numerical aperture of the light exiting the pinhole 108 and the numerical aperture NAMS of the microscope would reduce the amount of light reaching the sample to be imaged. Alternatively, the pinhole diameter DP could be increased to allow more light through the pinhole 108 and more efficient light transmission to the sample without changing the magnification M, but this would result in a larger spot size and lower resolution. Thus, the use of a non-single mode fiber in a single point scanning confocal microscope would require either a reduction in the system resolution, a reduction in light transmission to the sample, or a combination of both.
A recent development in optical microscopy has been the parallel application of the confocal technique. By the use of various optical means, a plurality of near-diffraction-limited illumination points are projected onto or into the sample. Each of these points is imaged through a corresponding pinhole at a conjugate focal plane onto an image sensor of a detector, such as a high-sensitivity imaging camera. In effect, such a system operates as a plurality of point scanning confocal systems operating in parallel. Several commercial implementations of this concept exist on the market today and can be referred to in general as multiplexed confocal systems.
One implementation of a multiplexed confocal system uses a spinning disk comprising a pattern of several thousand pinholes. An example of one such spinning disk confocal system is one which comprises a Nipkow disk. The use of a multiplexed confocal system employing the Nipkow disk method with microlenses has been disclosed in, for example, U.S. Pat. No. 7,592,582 to Mikuriya et al. The microlenses create a plurality of focal points. A confocal system which creates multiple focal points using microlenses, micromirrors or other focusing elements may be referred to as a multi-focal confocal system and forms a subset of multiplexed confocal systems.
In the instrument described in U.S. Pat. No. 7,592,582, the exciting laser light is coupled to the incident end of an optical fiber by a condenser lens and is guided by the optical fiber to an inlet of a confocal scanner unit. A diverging beam of exciting light emitted from the distal end of the optical fiber is converted into a collimated beam by a collimating lens. The collimated beam falls on a disk with a microlens array that focuses excitation laser light onto a pinhole disk (Nipkow disk) mounted on the same axis in such a way that each lens focuses its light onto a corresponding pinhole. Multiple exciting light beams are converged to a sample by an objective lens. Fluorescence and/or scattered light and/or reflected light originating from the sample passes through the objective lens again, returns through the same pinholes and is reflected by a dichroic mirror positioned between the microlens disk and the Nipkow disk. The image is then focused onto an image sensor by a relay lens.
In such an apparatus, the Nipkow disk is co-rotated with the microlens disk at a constant speed, and the converged points of light on the sample are scanned with the pinholes moved by the rotation. A plane of the Nipkow disk, a plane to be observed in the sample, and an image sensor plane are arranged to be conjugate with each other optically. Therefore, an optically sectioned image, that is a confocal image of the sample, is formed on the image sensor. Such a system as described above is made by Yokogawa Electric Corporation of Japan and given designations such as CSU-10, CSU-21, CSU-22 and CSU-Xl.
Other implementations of multi-focal confocal systems using microlenses exist where the key differences are in the geometry of the microlens patterns and the scanning mechanisms for moving the microlenses and pinholes. An example of such a system is called the Infinity and is built by VisiTech International Ltd. of Sunderland, United Kingdom.
Illumination methods for multi-focal confocal systems are similar to traditional point scanning systems and, until very recently, have used single mode fibers. In this case, the microlenses image the distal end of the fiber to many parallel pinholes at or near the diffraction limit. As with confocal point scanning systems, the typical radiation source for multi-focal confocal systems is a laser or multiple lasers.
There are disadvantages to using single mode fibers for some applications. Systems using single mode fibers are, in practice, restricted to radiation sources that emit light with small etendue, such as lasers with good beam quality, for example, beam quality factor M2<1.2. Laser sources with good beam quality can be coupled to single mode fibers with coupling efficiencies of approximately 45% to 85%, although the efficiency in practice is sometimes less. Lasers with lesser beam quality couple with even lower efficiencies. Single mode fibers can only operate as such over a limited spectral range. Above a given upper cutoff wavelength the fiber core is too small to transmit light with low losses. Below a lower cutoff wavelength, the light is no longer transmitted in a single mode. The Gaussian distribution of the single mode fiber output intensity is less than ideal for systems requiring even illumination. Only the central part of the Gaussian beam is often used, such that the variation in intensity is less than some amount, for example 20%. In such systems a compromise between uniformity in light distribution across an image plane and the light utilization efficiency is required because the peripheral part of the Gaussian beam is abandoned.
Another disadvantage of systems that use single mode fibers is the requirement for high thermal, mechanical, and temporal stability of the laser-to-fiber alignment and the high manufacturing cost of such stable systems. Designing a means of providing stable laser-to-fiber coupling, and the creation of systems coupling multiple lasers to a single mode fiber, can be challenging.
As an alternative to using single mode fibers for delivery of radiation in optical microscopes, the use of multimode fibers has recently been contemplated. A multimode fiber is an optical fiber that is designed to carry multiple light ray paths or modes concurrently over a broad spectrum of wavelengths. It can be thought of simply as a long light tube. The use of a multimode fiber may reduce the sensitivity of the coupling between the radiation source and fiber to mechanical and temperature influences, thereby enabling a variety of radiation sources and wavelengths to be used for illumination in an optical microscope.
In “A Mercury Arc Lamp-Based Multi-Color Confocal Real Time Imaging System for Cellular Structure and Function”, Cell Structure and Function, vol. 3, pages 133-141, 2008), Saito et al. describes the use of a multimode fiber with a 1 mm core diameter to couple an arc lamp to a Yokogawa CSU-10. The efficiency of the light coupled from the end of the multimode fiber through the CSU is reported to be 1%. While it was not clearly defined how this measurement was made, this number represents a low efficiency of light utilization. Saito et al. do not use this fiber with a laser but only with a broadband arc lamp source. Furthermore, with the use of such a large-diameter fiber, much of the lost light is scattered from the back surface of the pinhole disk, thus leading to a higher potential for a loss of contrast.
Use of a multimode fiber to efficiently deliver light emitted from a radiation source to a multi-focal confocal microscope has been disclosed by Berman in U.S. Patent Publication 2010/0142041. Berman discloses a method of selecting a core diameter and a numerical aperture of a multimode fiber such that light emitted from a distal end of the multimode fiber is transmitted through the confocal pinhole array with reasonable efficiency.
Typically, the intensity of light emitted from a distal end of a multimode fiber decreases at points further from the optical axis of a multimode fiber in the transverse plane. Therefore, a trade-off is made between light utilization efficiency and uniformity of illumination of the microscope sample. This trade-off may be realized by limiting the sample illumination to light from the central area of the collimated beam. Illuminating a smaller area may result in more uniform illumination but may use a smaller fraction of the light from the multimode fiber. Illuminating a larger area may result in better light utilization efficiency but may reduce the uniformity of the illumination.